芽孢杆菌(Bacillus subtilis No.16A)苎麻脱胶聚半乳糖醛酸裂解酶的纯化及酶学性质

通过丙酮沉淀,阳离子交换层析,凝胶过滤,从苎麻脱胶菌Bacillus subtilis No.16A发酵液中纯化了聚半乳糖醛酸酶（Pgase）.结果表明该酶分子量为30.2ku,最适作用pH为9.0,最适作用温度为50℃,在55℃以下、中性pH（6.0～8.0）范围内稳定, Ca^2＋、Mg2＋对该酶有激活作用, Cu^2＋、Mn^2＋、Co^2＋、Hg^2＋有抑制作用,酶解产物在235 nm处有强吸收峰,说明该酶是聚半乳糖醛酸裂解酶.

On the Bacteriostatic Activity of Bacillus subtilis and Pyraclostrobin as Well as Their Mixtures to Grape Anthracnose and the Field Disease Control Efficiency

[Objective] To screen out the biological compound bactericides for grape anthracnose, reduce and replace the use of chemical pesticide. [Methods] The de- termination on the indoor bacteriostatic activity of different proportions of Bacillus subtilis and pyraclostrobin to grape anthracnose was carried out, and mycelial growth rate method was adopted to determine the toxicity of Bacillus subtilis and pyraclostrobin as well as their 5 mixtures to grape anthracnose. [Results] The EC50 of Bacillus subtilis and pyraclostrobin as well as their mixture combinations of 1：1, 1：2, 1：3, 1：4 and 1：5 to grape anthracnose were respectively 1.969 8, 1.527 4, 1.373 2, 1.294 8 and 1.247 3 μg/ml; the synergistic coefficients （SR） of the 5 mix- ture combinations to grape anthracnose were 1.70, 1.25, 1.13, 1.12 and 1.12, re- spectively, in which the synergistic effect of 1：1 was the largest. The indoor biologi- cal activity of pyraclostrobin（EC50 was 1.054 0μg/ml） was higher than that of Bacil- lus subtilis（EC50 was 15.017 5 μg/ml）. 50 d after the agentia（before the harvesting）, the investigation results showed that 1 000-fold dilution, 1 500-fold dilution and 2 000- fold dilution as well as each single dosage of 20% pyraclostrobin .200×10^8 cfu/g Bacillus subtili wettable powder all had better control efficiency to grape anthracnose after fruit setting and before bagging, in which the treatments of high concentration and middle concentration were higher than the treatments of low concentration and two single dosages： the highest control efficiency of high concentration was 90.03%, which was higher than all other treatments; the control efficiency of middle concen- tration was 87.01%, which was higher than that of low concentration and each sin- gle dosage; the control efficiency of low concentration was 84.11%, which was high- er than 1 000-fold dilution of 1 000×10^8 cfu/g Bacillus subti/i wettable powder （the control efficiency was 64.60%） and 2 000-fold dilution of 250 g/L Bacillus subti/i wettable powder （the control efficiency was 81.07%）. In addition, each treatment al- so had better control efficiency to other cluster diseases, such as white rot, etc., and the control efficiency was almost the same as that of anthracnose. [Conclusion] It was suggested that the prevention concentration of 20% pyraclostrobin .200×10^8 cfu/g Bacillus subtili wettable powder to grape anthracnose after fruit setting and before bagging was 1 000-fold - 2 000-fold dilution.

转枯草芽孢杆菌纤溶酶(Bacillus subtilis fibrinolytic enzyme,BSFE)基因对烟草生长发育的影响

将枯草芽孢杆菌纤溶酶(Bacillussubtilisfibrinolyticenzyme,BSFE)基因及其信号肽序列转入烟草,转基因烟草表现出不同于野生型烟草的生长特性,如营养器官生长速率下降、叶片数目减少、叶片狭长、叶片宽度/叶片长度值降低以及植株矮小、节间缩短等。推测其与外源BSFE的蛋白水解活性有关。

生物炭载Bacillus subtilis菌粒对连作芹菜生长的影响

为解决土壤连作障碍对蔬菜生长发育的不利影响,本研究以连作3年的芹菜土壤为研究对象,采用随机区组试验,设置5种处理:单独施加生物炭(BC)、单独施加Bacillus subtilis菌剂(BS)、生物炭与菌剂混合施加(BB)、施加生物炭载B.subtilis菌粒(BP)、不施加生物炭与菌剂(CK),盆栽种植芹菜,测定其生长与品质指标,探究生物炭和B.subtilis对连作芹菜生长的影响。结果表明,BB、BP处理组芹菜的生长和品质指标都高于其他土壤处理组,BP处理组的芹菜鲜质量、可溶性蛋白含量以及纤维素含量均显著高于BB处理组,分别为60.25 g/棵、27.25 mg/g、2.48%,其他指标两处理间无显著差异。不同土壤处理组芹菜的生长和品质指标都高于CK处理,其中BP处理组各指标值最高,尤其是芹菜鲜质量,增幅高达226.56%。因此,施加生物炭载B.subtilis菌粒提高了连作芹菜生长指标与品质,为农业生产上缓解蔬菜连作障碍奠定了理论基础。

氨肽酶生产菌株Bacillus subtilis LBJ4-5的全基因组测序及基因功能分析

本研究从郫县豆瓣中分离获得一株高产氨肽酶菌株B. subtilis LBJ4-5,通过溶血性试验及抗生素敏感性试验评估了其安全性。同时,采用Illumina二代测序技术和第三代高通量Pacbio测序平台对B. subtilis LBJ4-5进行全基因组测序,获得其基因组特征信息,并通过COG、GO、KEGG、CAZY、CARD和VFDB等数据库进行基因预测及功能注释,以及评价其氨肽酶生产性能和安全性。结果显示,B. subtilis LBJ4-5经48 h摇瓶培养后发酵上清中的氨肽酶活达到179.23 U/mL,菌株无溶血活性且对头孢唑林、万古霉素、青霉素G、头孢氨苄等10种抗生素均高度敏感(直径≥20 mm),显示出良好的安全性。B. subtilis LBJ4-5的基因组由一条环状闭合DNA及一个环状质粒组成,大小为4053926 bp,GC含量为43.66%,共预测到4285个蛋白质编码基因、85个tRNA基因、33个rRNA基因、119个sRNA。在COG、GO、KEGG、CAZY、CARD和VFDB数据库中分别注释到3386、2581、2552、144、256和401个功能基因。进一步的信息挖掘分析表明,B. subtilis LBJ4-5的基因组中含有31个氨肽酶编码基因。而毒力基因与耐药性基因的预测分析,进一步证实了B. subtilis LBJ4-5菌株的安全性。综上,B.subtilis LBJ4-5可用作发酵食品加工的发酵菌剂。

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Spontaneous cerebral abscess due to Bacillus subtilis in an immunocompetent male patient: A case report and review of literature

BACKGROUND Bacillus subtilis(B. subtilis) is considered a nonpathogenic microorganism of the genus Bacillus and a common laboratory contaminant. Only scarce reports of B. subtilis central nervous system infection have been reported, mainly in the form of pyogenic meningitis,usually in cases of direct inoculation by trauma or iatrogenically.CASE SUMMARY A 51-year-old man, with a free previous medical history, presented to the Emergency Department of our hospital complaining of recurrent episodes of left upper limb weakness, during the last month, which had been worsened the last 48 h. During his presentation in Emergency Department he experienced a generalized tonic-clonic grand mal seizure. Brain magnetic resonance imaging(MRI) scan with intravenous Gadolinium revealed a 3.3 cm × 2.7 cm lesion at the right parietal lobe surrounded by mild vasogenic edema, which included the posterior central gyrus. The core of the lesion showed relatively homogenous restricted diffusion. Post Gadolinium T1 W1 image, revealed a ring-shaped enhancement. Due to the imaging findings, brain abscess was our primary consideration. Detailed examination for clinical signs of infectious foci revealed only poor oral hygiene with severe tooth decay and periodontal disease, but without detection of dental abscess. The patient underwent surgical treatment with right parietal craniotomy and total excision of the lesion. Pus and capsule tissue grew B. subtilis and according to antibiogram intravenous ceftriaxone 2 g bids was administered for 4 wk. The patient remained asymptomatic and follow-up MRI scan two months after operation showed complete removal of the abscess.CONCLUSION This case highlights the ultimate importance of appropriate oral hygiene and dental care to avoid potentially serious infectious complications and second, B. subtilis should not be considered merely as laboratory contaminant especially when cultivated by appropriate central nervous system specimen.

Degradation of glucosinolates in rapeseed meal by Lactobacillus delbrueckii and Bacillus subtilis

Lactobacillus delbrueckii and Bacillus subtilis were employed as a new combination of strains to treat rapeseed meal by solid-state fermentation,aiming to efficiently degrade the glucosinolates,which are the main toxin in the meal.Single-factor tests and Response surface methodology(RSM)were used to optimize the fermentation parameters.Under the optimum fermentation parameters of 15%total injection volume of the mixture of Lactobacillus delbrueckii and Bacillus subtilis with a ratio of 2:1,bran content of16%,feed to water ratio of 1:1.5,fermentation temperature of 36°C and fermentation time of 72 h,the content of glucosinolates in rapeseed meal was decreased from 64.558μmol/g to 3.473μmol/g,reaching a high degradation rate(94.62%).The high detoxification rate by a consortium of Lactobacillus delbrueckii and Bacillus subtilis provides a bright application prospect in feed utilization of rapeseed meal.

Research Progress of Antimicrobial Peptides and the Potential Applications of Bacillus subtilis

This paper presents a comprehensive account of antimicrobial peptides (AMPs) derived from various sources, including animal, plant, and microbial origins, along with an examination of their structural characteristics and biological activities. Specifically, the potential of Bacillus subtilis as a safe and effective host for the production of AMPs is discussed. B. subtilis exhibits a notable capacity for protein secretion and is also capable of efficiently producing AMPs without the presence of endotoxin contamination. The research indicates that the production efficiency of AMPs derived from B. subtilis can be significantly enhanced through the application of genetic engineering and synthetic biology techniques. This advancement holds considerable potential for applications in food preservation, agriculture, medicine, and various other fields. The paper additionally investigates the stability of AMPs under diverse conditions of temperature, pH, and enzymatic treatment, and highlights the necessity for further research to facilitate the advancement of these AMPs for practical applications.

Postharvest Biological Control of Apple Anthracnose by Bacillus subtilis BS80-6,A Mutant Strain through Ion Implantation

The original Bacillus subtilis was isolated from the soil of an apple orchard,and B. subtilis strain BS80-6,selected from Bacillus subtilis by ion implantation,was used as an antagonist for postharvest biological control against apple anthracnose（ colletotrichum gloeosporioides）. The mechanisms of action and efficacy of Bacillus subtilis strain BS80-6 against apple anthracnose caused by colletotrichum gloeosporioides were studied in vitro and on apples in controlled and semi-commercial conditions. An application of cell suspension（ 108 cells per mL） of the antagonist in artificial wounds of apples reduced growth of C. gloeosporioides after storage at different temperatures. The inhibitory actions of cell culture to hyphal growth and to spores germination were 86. 6% and 98. 65%,respectively,and the control efficacy of cell culture against the disease was 60. 34% at room temperature. The results showed that BS80-6 was more effective against apple anthracnose.The cell culture of BS80-6 received better control efficacy against apple anthracnose than culture filtrates and autoclaved cell. All treatments significantly inhibited the disease both in vitro and in vivo compared with control. Fruits treated with cell culture and stored at 10 ℃ had lower infection rate,more delayed formation of acervwlus and smaller lesion diameter than those stored at 20 ℃. There was better control efficacy in fruits inoculated with spores after application of BS80-6. The mechanisms of BS80-6 activity appeared to involve production of an antifungal substance,deformation of hyphal cell and disintegration of cell wall. Besides,BS80-6 could improve the activity of major defense enzymes of apple,such as peroxidase（ POD） and polyphenol oxidase（ PPO）.

Fermentation Production of Ganoderma lucidum by Bacillus subtilis Ameliorated Ceftriaxone-induced Intestinal Dysbiosis and Improved Intestinal Mucosal Barrier Function in Mice

Objective This study aimed to develop a type of Ganoderma lucidum(G.lucidum)-probiotic fermentation broth that can effectively improve the intestinal mucosal barrier function of mice with ceftriaxone-induced intestinal dysbiosis.Methods By means of absorbance of optical density(OD)value and phenol-concentrated sulfuric acid measurement of polysaccharide content,the probiotic species can grow on the medium of G.lucidum were screened out,and the concentration of the medium of G.lucidum was determined,and the fermentation broth was prepared for subsequent experiments.Thirty-two SPF grade male BALB/c mice were randomly divided into four groups(eight mice in each group),namely control group(CON),intestinal mucosal barrier damage model group(CS),fermentation broth intervention group(FT)and G.lucidum medium intervention group(GL),respectively.The intestinal dysregulation model was induced by intra-gastric administration of 0.2 mL ceftriaxone sodium(twice a day for seven consecutive days).From day 8,the FT group and GL group were gavage with 0.2 mL fermentation broth and G.lucidum medium,respectively.On day 15,all mice were sacrificed.To draw the weight curve and measure the cecal index;pathological examination of colon tissues with HE staining;serum levels of LPS,IL-10,TNF and IL-6 were detected by ELISA.Flow cytometry was used to analyze the changes of CD3+T cells,CD4+T cells,CD8+T cells and macrophages in spleen.16S rRNA sequencing was performed to detect the intestinal microbiota structure of mice.Results Bacillus subtilis can decompose and utilize G.lucidum fruiting body medium,and the suitable concentration of G.lucidum fruiting body medium is 33.2 mg/mL.The effect of Bacillus subtilis-G.lucidum fermentation broth on the damage of intestinal mucosal barrier caused by ceftriaxone sodium was reduced,the body weight of mice recovered and colon swelling improved,colon histopathological injury was alleviated,inflammatory cell infiltration was alleviated,serum IL-10 increased significantly,LPS、TNF-αand IL-6 decreased significantly compared with model group,and the proportion of T cells and intestinal dysbiosis was improved.Conclusions The experimental results suggest that Bacillus subtilis-G.lucidum fermentation broth can effectively improve the intestinal barrier function damage,immune dysfunction and intestinal dysbiosis caused by antibiotic overdose,and has a certain regulatory effect on intestinal mucosal barrier function.

Bacillus subtilis 168中亚硝酸盐还原酶基因的敲除及表型鉴定

该研究首先通过同源重组方式将Bacillus subtilis 168菌株nir基因位点置换为两端具有同向排列的loxP序列选择性标记基因,再运用Cre重组酶将两个loxP间的全部序列切除,成功敲除了Bacillus subtilis 168菌株亚硝酸盐还原酶(nitrite reductase,NiR)的3个亚基基因nasB、nasD和nasE。其中,敲除nasD基因的B.subtilis 168菌株在不同生长时期均未分解培养基中亚硝酸盐,表明该nasD基因缺陷型菌株NiR活性已被完全消除,不再具备降解亚硝酸盐的能力。

Physicochemical Properties and Fibrinolytic Activity of Ginseng Powder Fermented with <i>Bacillus subtilis</i>Isolated from <i>Cheonggukjang</i>

In order to obtain all the properties of fermented ginseng, we fermented ginseng using Bacillus subtilis (B. subtilis) isolated from Cheonggukjang. A sterilized ginseng medium was made with 4-year-old ginseng powder and distilled water (300% ginseng powder [w/w]), and the ginseng was fermented by B. subtilis (1% ginseng medium) followed by incubation at 37°C for 3, 5, 7, and 10 days. The growth of B. subtilis in the ginseng medium significantly increased up to 9 log CFU/g, but no significant difference was observed after 3 days. As the fermentation progressed, the ginsenoside Rd and Rg+Rh1 contents increased by 255.3% - 322.5% and 165.6% - 228.6%, respectively, whereas the Rc, Re, and Rg1 contents decreased by 30.7% - 39.6%, 10.5% - 12.8%, and 16.2% - 16.6%, respectively. After 3 days of fermentation, a 6.25% - 7.12% viscous substance was produced;thereafter, the viscous substance was gradually reduced until it disappeared. The viscosity of the medium significantly decreased with a longer fermentation time. Fibrinolytic activity increased during 3 - 10 days of fermentation, indicating a relative activity of 85.0% - 100.0%.

Bacillus subtilis fmbJ脂肽类抗菌物质的分离和鉴定

对BacillussubtilisfmbJ脂肽类抗菌物质的分离和鉴定进行了系统研究。通过HPLC层析确定BacillussubtilisfmbJ抗菌物质由多种组分构成,其中含有保留时间与surfactin相似的成分。通过TLC层析和原位酸解确定BacillussubtilisfmbJ抗菌物质含有两个具有闭合肽键类的物质,其中之一为迁移率Rf与标样surfactin非常相近的组分。通过ESI-MS分析检测到BacillussubtilisfmbJ抗菌物质含有分子量与fengicin相同的m/z1449.9、m/z1463.8、m/z1477.8、m/z1491.9和m/z1505.9五种同系物,和分子量与surfactin相同的m/z1008.8、m/z1022.8和m/z1036.8三种同系物。

Bacillus subtilis fmbR抗菌物质的分离和鉴定

【目的】对Bacillus subtilis fmbR产生的抗菌物质进行分离和鉴定研究,以确定抗菌物质的组成和结构。【方法】采用HPLC和TLC层析对Bacillus subtilis fmbR抗菌物质进行分离纯化,通过ESI-MS和MALDI-MS分析对抗菌物质的组成和结构进行初步鉴定。【结果】HPLC层析表明了Bacillus subtilis fmbR抗菌物质含有保留时间与surfactin相似的成分。TLC层析和原位酸解证明了Bacillus subtilis fmbR抗菌物质含有闭合肽键类的物质,其中之一为相对迁移率Rf与标样surfactin相近的组分。采用ESI-MS分析检测到Bacillus subtilis fmbR抗菌物质含有分子量与surfactinA相同的m/z1009.1、m/z1023.2和m/z1037.0等3种同系物;通过MALDI-MS分析获得[M+H]+为m/z 3403.95抗菌物质,该物质分子量与Bacillus subtilis 168产生的细菌素subtilosin的m/z3403.3相同。【结论】Bacillus subtilis fmbR抗菌物质由C13～C15的3种surfactinA同系物和一种羊毛硫抗生素subtilosin组成。

Screening for the Strain Highly Producing Antagonistic Substance from Bacillus subtilis B47 by UV Mutagenesis

[Objective] The aim of this study was to breed new strains which have higher inhibitory effects on the pathogens of watermelon fusarium wilt.[Method] The endophytic Bacillus subtilis B47 strain was obtained from tomato stems by UV mutagenesis for two consecutive times,then genetic stability as well as physiological and biochemical properties of mutant strains were studied.[Result] The antibacterial activity of all the three mutant strains F303,F304 and F305 was higher than that of B74 strain.After subculture of 10 successive generations,the antibacterial activity of all the three mutant strains for the pathogens of watermelon fusarium wilt decreased,but the antibacterial activity of F305 strain decreased the least,indicating its best genetic stability among the tested strains.The antibacterial circle diameter of F305 strain was 5 mm larger than that of wild strain B47 under the same condition.The mutant strain F305 was in logarithmic growth phase within 36 h and in stationary phase within 36-96 h,while its optimum growth temperature was 35 ℃.F305 strain could grow in sodium salt with the concentration of 1%-10%,but it grew best at the concentration of 1%.Physiological and biochemical responses of F305 strain were in accordance with those of wild strain B47.[Conclusion] This study lays the foundation for the factorial production of antagonistic substance by B47 strain and new methods of preventing from the pathogens watermelon fusarium wilt.

Bacillus subtilis B2产1-脱氧野尻霉素(DNJ)发酵条件优化

为探索制备含有1-脱氧野尻霉素(1-deoxynojimycin,DNJ)的降血糖功能性食品的新方法,以产DNJ的菌株Bacillus subtilisB2为初始菌株,以价廉的农产品加工副产物豆渣为原料,通过对发酵条件的优化,获得富含功能因子DNJ的发酵液。结果表明:接种量对α-葡萄糖苷酶抑制活性影响不明显,当接种量大于104个/mL时,发酵液的α-葡萄糖苷酶抑制活性均在20以上。而豆渣浓度、发酵温度、培养基初始pH值及发酵时间对发酵液α-葡萄糖苷酶抑制活性影响较大,在豆渣质量浓度30mg/mL,初始pH值6～8,发酵温度40℃的条件下发酵48h,发酵液表现出较高的α-葡萄糖苷酶抑制活性。以Bacillus subtilisB2发酵豆渣的发酵液为原料开发含DNJ的降糖功能食品,可以大大提高豆渣的利用价值,为降糖功能食品的开发利用开辟新途径。

枯草芽孢杆菌(Bacillus subtilis)B-912对采后柑桔果实青、绿霉病的抑制效果

用从土壤中分离到的枯草芽孢杆菌 B- 912作为生防拮抗菌 ,主要研究在不同贮藏温度下不同接种时间的 B- 912活菌液、热处理液和过滤液对柑桔青、绿霉病的抑制效果。B- 912活菌液的抑菌效果最佳 ,热处理液和过滤液次之 ,但均显著地好于对照。较高的贮藏温度有利于拮抗菌对病菌的抑制作用 ,活菌液处理的果实在 2 5℃下的发病率及病斑直径均小于 15℃下的果实 ,2 4 h后接种病菌孢子的果实其发病率及病斑直径一般都高于 4 8h后接种的果实。实验证明 ,枯草芽孢杆菌的抑菌机理主要是产生抗菌素。

脂肪酶产生菌Bacillus subtilis FS321的分离鉴定及其产酶条件的初步优化

从北方堆肥试样中分离筛选得到1株中度耐热脂肪酶产生菌,编号为FS321。对该菌株进行产酶条件的初步优化,得到了摇瓶发酵条件:产酶培养基M4,发酵周期为48～60 h,摇瓶发酵温度为37℃,通气量为25 mL/250 mL锥形瓶。所产脂肪酶在50℃,pH 9.0时表现最高活性。为了鉴定该菌株,分析该菌的细胞脂肪酸谱,克隆测序了其16S rDNA基因序列,系统进化树及细胞脂肪酸组分分析均表明,FS321与Bacillus subtilis具有最紧密的亲缘关系。

辣椒内生枯草芽孢杆菌(Bacillus subtilis)BS-2和BS-1防治辣椒炭疽病研究

辣椒内生枯草芽孢杆菌(Bacillus subtilis)BS-莉和BS-1,对胶孢炭疽菌(Colletotrichum gloeosporioides)引起的辣椒苗和果炭疽病有良好的防治效果,菌株培养液对苗炭疽病的防效分别为81.5％～93.3％和66.1％～79.2％;对果炭疽病的防效分别为80.0％～100％和60.0％～100％,喷施菌株培养液后间隔24h以上接种病原菌的防病效果比两者同时接种的高。菌株外分泌物对病菌菌丝生长、分生孢子产生和萌发及附着胞形成均有明显的抑制作用。菌株菌体及其胞外分泌物均有防病作用,菌体可能起主导作用。另外,2菌株培养液浸种,对辣椒还有明显的促生效果。BS-2菌株的防病和促生效果均比BS-1高。